Abstract
Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.
Highlights
Dynamin is a large, self-assembling GTPase required for clathrin-mediated endocytosis (CME)
Our analyses of two distinct classes of dynamin GTPase mutants helps to resolve the different roles of GTP binding, basal and assembly-stimulated GTP hydrolysis in dynamin function during clathrin-mediated endocytosis and for catalyzing membrane fission
A major finding of these studies is that, compared to assemblystimulated GTPase activity resulting from dimerization of G domains, fission activity of a fixed number of WT dynamin molecules is more sensitive to inhibition by both S45N and Q40E mutants
Summary
Self-assembling GTPase required for clathrin-mediated endocytosis (CME). An additional burst of recruitment is observed at later stages of CME [4,5,6], which presumably reflects the self-assembly of dynamin into collarlike structures at the necks of deeply-invaginated CCPs to mediate membrane fission and clathrin coated vesicle release. Functional analysis of dynamin mutations has provided significant mechanistic insight into its role in CME (reviewed by [10,11,12]) These studies have revealed that dynamin-mediated membrane fission requires GTP binding and hydrolysis [13,14,15], GTP-driven conformational changes [15,16], membrane binding [17,18,19], self-assembly [20,21] and curvature generation through shallow insertions into the lipid bilayer [22]. Dynamin has been extensively studied over the past 20 years, there remains considerable controversy and uncertainty as to its exact role(s) in CME and the mechanism of dynamin-catalyzed membrane fission [8,10,23]
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